The aim of the proposed research is to design contrast agents that can measure local levels of soluble Vascular Endothelial Growth Factor (VEGF) via safe, noninvasive means. VEGF helps to form new blood vessels that feed a growing tumor, and thus elevated VEGF levels in the tumor environment correlates negatively with survival in breast and other cancers. Patients who have received a positive result from a cancer screening usually undergo an invasive biopsy to measure the levels of such biomarkers. However, high false positive rates in screening have led to unnecessary biopsies that burden a patient with physical, financial, and psychological costs. Thus it would be ideal to measure biomarker levels in a specific environment - where a specific biomarker may be secreted, overexpressed, or recruited by receptor ligands - through a highly sensitive and specific noninvasive technique. The PIs plan to create such a technique by leveraging their design of stimulus-responsive ultrasound contrasts agents with their experience in using ultrasound for contrast-enhanced cancer imaging. Microbubbles are potent ultrasound contrast agents because ultrasound causes the bubble to expand and contract, generating a nonlinear echo that can be detected with excellent specificity. Biomarker-sensitive activity will be imparted to the VEGF-sensitive microbubbles through placement of aptamers, or DNA sequences with affinity for proteins, around the bubble to form a network of links that prevent the microbubbles from fluctuating in size. VEGF will cause removal of the aptamer strands, restoring shell flexibility an full contrast activity. In previous research, the PI team successfully employed a similar strategy to create microbubbles that exhibited sensitivity to elevated thrombin levels for detection of deep venous thrombosis. In addition, tumor-specific targeting ligands will be added to hold microbubbles at the tumor site, ensuring sufficient signal. Second, the microbubbles will be dually imaged such that comparison of these received signals will provide a more accurate, quantitative measurement of VEGF. Completion of the proposed research will be accomplished through satisfaction of the following specific aims: (1) microbubbles with sensitivity to VEGF will be designed and validated in nonbiological phantoms, (2) new imaging techniques will be designed for microbubble-based quantification of VEGF levels through establishment of signal-pressure relationships, and (3) VEGF will be quantified in breast cancer cell lines in vitro and an in vivo breast cancer xenograft model. The long-term goal is to apply this technology to other types of cancers, including ovarian and pancreatic, and advance these tools for use in clinical oncology.